Objective To investigate the effect and potential mechanism of cuttlebone (CB)/bovine serum albumin (BSA) photocrosslinked hydrogel on rats with critical sized bone defects (CSBD) model in skull tissues. Methods SD rats were randomly divided into control group, positive control group, BSA group, 0.5% CB/BSA group, 1.5% CB/BSA group and 3.0% CB/BSA group, with six rats in each group. A rat CSBD model was established using a 5 mm outer diameter ring drill, while the control group only underwent modeling without implants, positive control group implanted with Bio-Oss^® collagen bone filling material, CB/BSA photocrosslinked hydrogels with CB mass fraction of 0, 0.5%, 1.5% and 3% were implanted in BSA group, 0.5% CB/BSA group, 1.5% CB/BSA group and 3.0% CB/BSA group, respectively. After eight weeks after surgery, the microstructural changes of bone tissue were analyzed using micro computed tomography (Micro CT). Hematoxylin-eosin (HE) and Masson staining were used to observe pathological changes in skull tissue. Immunohistochemistry and immunofluorescence were used to detect the positive expressions and fluorescence intensity of collagen type I alpha 1 (COL1A1), platelet endothelial cell adhesion molecule-1 (PECAM-1) and osteocalcin (OCN) in skull tissue, respectively. qRT-PCR and Western blotting were used to detect the gene and protein expressions of Wnt family members 3A (WNT3A), β-catenin, vascular endothelial growth factor A (VEGFA), COL1A1, PECAM-1 and OCN in skull tissue, respectively. In order to evaluate the safety of hydrogel in vivo, the abdominal aorta blood of rats was collected to analyze the changes of blood routine and serum liver and kidney function indexes. Results Micro-CT results showed that no new bone formation was observed in the bone defect area of rats in control group, indicating that bone healing was not achieved during the observation period. The pathological tissue section results showed that the defect area in control group was mainly composed of loose fibrous tissue. In CB/BSA photocrosslinked hydrogel group, new bone formation was observed. Compared with control group, bone volume/tissue volume (BV/TV), bone mineral density (BMD and trabecular thickness (Tb.Th) of rats in CB/BSA photocrosslinked hydrogel group were significantly increased (P < 0.05), bone surface/bone volume (BS/BV) was significantly decreased (P < 0.05), the positive expressions and fluorescence intensity of COL1A1, PECAM-1, OCN in skull tissue were significantly increased (P < 0.05), and the expression levels of COL1A1, PECAM-1, β-catenin, WNT3A, VEGFA, OCN were significantly increased (P < 0.05), among which, the 3.0% CB/BSA photocrosslinked hydrogel group had the best comprehensive effect. No significant abnormalities were found in the blood routine and serum liver and kidney function indicators of rats in each group, and no pathological changes such as inflammation, necrosis or fibrosis were observed in the main organs. Conclusion CB/BSA photocrosslinked hydrogel shows good bone repair effect and safety in vivo, and its osteogenic effect may be mediated by activation of Wnt/β-catenin signaling pathway.

cuttlebone  /  bovine serum albumin  /  photocrosslinked hydrogel  /  bone repair  /  Wnt/β-catenin signaling pathway